Single-cell genomic methods to generate ex vivo cell systems that recapitulate in vivo biology with improved fidelity

A method of generating an in vitro cell-based system that faithfully recapitulates an in vivo phenotype of interest comprising:

• a) determining, using single cell RNA sequencing, gene expression for single cells in an initial in vitro cell-based system to computationally identify cell clusters of enteroendocrine Paneth cell types, wherein the initial in vitro cell-based system is an in vitro intestinal organoid cell-based system comprising Paneth cells, wherein the organoid cell-based system is obtained from an intestinal stem cell enriched organoid produced by enriching for murine LGR5+ intestinal stem cells in a scaffold and medium containing growth factors EGF (E), Noggin (N), R-spondin I (R), CHIR99021 (C), and valproic acid (V);
• b) identifying differences in the gene expression for single Paneth cells in the initial in vitro cell-based system by performing differential gene expression analysis for clusters of single Paneth enteroendocrine cell types and Paneth cells in the in vivo system having the phenotype of interest, wherein differential gene expression analysis comprises comparing a gene expression distribution as determined by single cell RNA sequencing of the initial in vitro cell-based system and a gene expression distribution as determined by single cell RNA sequencing in the Paneth cells in vivo system;
• c) identifying differential gene expression for Wnt and Notch pathways for Paneth cells identified in the initial in vitro cell-based system and the Paneth cell in vivo system in step (b);
• and
• d) modulating Wnt and/or Notch signaling in the initial in vitro cell-based system with one or more agents comprising a Wnt signaling activator and Notch signaling inhibitor to induce a shift that reduces the differences in gene expression for the Paneth cells between the initial in vitro cell-based system and the Paneth cell in vivo system, thereby generating the in vitro cell-based system that faithfully recapitulates the in vivo phenotype of interest,
• wherein the differential gene expression analysis comprises measuring a Euclidean distance, Pearson coefficient, Spearman coefficient, or any combination thereof, and
• wherein the differential gene expression analysis comprises 10 or more genes, 20 or more genes, 30 or more genes, 40 or more genes, 50 or more genes, 100 or more genes, 500 or more genes, or 1000 or more genes;
• or wherein the differential gene expression analysis comprises one or more cell pathways;
• or wherein the differential gene expression analysis comprises a transcriptome of the Paneth cell in vivo system.